Peripherally-restricted dual-acting kappa and delta opioid agonist for analgesia in pain states involving the inflammatory response

ABSTRACT

The present disclosure teaches the use of a dual-acting opioid agonist for the treatment of pain (e.g., inflammatory pain). The opioid agonist activates both the kappa and delta opioid receptors to provide synergistic reduction in pain. The opioid agonist is peripherally restricted and does not cross the blood-brain barrier.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and benefit of, U.S. ProvisionalPatent Application No. 62/529,285 filed Jul. 6, 2017, the contents ofwhich are hereby incorporated by reference in their entirety.

FIELD OF THE DISCLOSURE

The present disclosure teaches the use of a dual-acting opioid agonistfor the treatment of pain (e.g., inflammatory pain). The opioidactivates both the kappa and delta opioid receptors to providesynergistic reduction in pain, dual agonism.

BACKGROUND OF THE DISCLOSURE

Opioid analgesics can be useful analgesics for the treatment of pain.These drugs, such as heroin and morphine, are agonists at mu opioidreceptors (MORs) in the central nervous system. However, because thesedrugs can cross the blood-brain barrier to the central nervous system,their use can cause unwanted side effects such as addiction. Moreover,because not all pain is mediated by MORs, these drugs may also be onlypartially effective for the treatment of certain types of pain.Accordingly, there is a need for safe and effective analgesics for thetreatment of pain (e.g., chronic and/or inflammatory pain) that do notsuffer from the side effects of traditional opioids such as morphine.

According to the National Institute on Drug Abuse, more than 115 peopledied every day in 2017 after overdosing on opioids. The total economiccost of opioid misuse in the United States is $78.5 billion per year.The opioid crisis has been caused and/or exacerbated byover-prescription of opioid pain-relievers for the treatment of pain(Soelberg et al., Antesth & Analg. 2017; 125(5): 1675-1681).Specifically, prescription opioids can be highly addictive and arewidely misused. Indeed, all m-activating opioids including heroin,morphine, and many other commonly prescribed opioids for pain arepotentially addictive (Ostling et al., Curr Pain Headache Rep.2018:22(5):32).

Despite their widespread use, many prescription opioids are poorlyeffective for certain types of pain such as inflammatory and/or chronicpain. For example, pain due to bone cancer can be only partiallyresponsive to prescription opioids such as morphine that targetmu-opioid receptors (MOR). Without wishing to be bound by theory, thisis likely because MOR can be down-regulated in bone cancer and thustargeting MOR can result in only a partial response (Yamamoto et al A.Neuroscience. 2008; 151(3):843-53). Moreover, in bone cancer, multipleother non-opioid pain pathways are active, including involvement ofinflammatory mediators of bradykinin, further limiting the effectivenessof treatments that only target MOR (Mantyh, P. Bone cancer pain: causes,consequences, and therapeutic opportunities. Pain. 2013;154(S1):S54-62).

Additionally, in neuropathic pain there is a shift away from mu-opioiddominated pathways to noradrenergic pathways (Bee et al. Pain. 2011;152(1) 131-9). Likewise, in fibromyalgia there is a reduced central MORavailability (Harris el al. J Neurosci. 2007; 12; 27(37):10000-6). Thereduced activity of MOR in these and other types of pain can thus reducethe effectiveness of drugs such as traditional opioids that only targetMOR (e.g., morphine).

Moreover, in all chronic pain states, mu-opioid agonists can themselvesinduce microglial activation that can in turn induce hyperalgesia, alowered pain threshold, and a primed microglial phenotype that persistseven after opioid discontinuation. This can worsen rather than alleviatechronic pain even after opioid discontinuation (Merighi et al. BiochemPharmacol. 2013; 86(4): 487-96). Thus, in some cases patients sufferingfrom chronic pain may realize incomplete relief when using traditionalopioids such as morphine, even despite increasing doses. This cycle ofincreasing dosage without adequate pain relief can result in dependenceand addiction.

On the other hand, due to concerns over addiction and overdose, otherswho experience chronic pain may suffer undertreatment (Reville et al.Ann Palliat Med. 2014; 3(3):129-38). For instance, in many parts of thedeveloping world, access to opioids even for acute pain and/or cancerpain can be restricted due to concerns over addiction and overdoseoutlined above (Id). Even in the United States, some patients can sufferfrom an undertreatment of pain. For example, patients with cognitiveimpairment and the elderly can be especially susceptible to the centralnervous system effects of traditional opioids such as morphine and insome cases are not prescribed enough to meet their pain management needs(American Geriatrics Panel on the Pharmacological Management ofPersistent Pain in Older Persons. Pain Med. 2009; 10.1062-1083).Furthermore, alternative effective analgesics are not available (Id).This represents a significant unmet medical need and is a significantpublic health crisis that does not receive adequate attention (Id).

Despite the unmet need for safe and effective pain relievers, no suchdrug is currently available. Accordingly, there is a need for a safe andeffective pain treatment that does not have the drawbacks associatedwith traditional opioid drugs.

BRIEF SUMMARY OF THE DISCLOSURE

One aspect of the present disclosure relates to a method of treatingpain in a subject in need thereof, the method comprising administeringto the subject a therapeutically effective amount of Compound 1:

or a pharmaceutically acceptable salt, prodrug, solvate, hydrate,tautomer, or isomer thereof.

Another aspect of the present disclosure relates to the use of theCompound 1:

or a pharmaceutically acceptable salt, prodrug solvate, hydrate,tautomer, or isomer thereof for the treatment of pain.

Another aspect of the present disclosure relates to the use of theCompound 1:

or a pharmaceutically acceptable salt, prodrug, solvate, hydrate,tautomer, or isomer thereof in the manufacture of a medicament for thetreatment of pain.

One aspect of the present disclosure relates to a method of preventingpain in a subject in need thereof, the method comprising administeringto the subject a therapeutically effective amount of Compound 1:

or a pharmaceutically acceptable salt, prodrug, solvate, hydrate,tautomer, or isomer thereof.

Another aspect of the present disclosure relates to the use of theCompound 1:

or a pharmaceutically acceptable salt, prodrug, solvate, hydrate,tautomer, or isomer thereof for the prevention of pain.

Another aspect of the present disclosure relates to the use of theCompound 1:

or a pharmaceutically acceptable salt, prodrug, solvate, hydrate,tautomer, or isomer thereof in the manufacture of a medicament for theprevention of pain.

Another aspect of the present disclosure relates to a pharmaceuticalcomposition comprising Compound 1:

and a pharmaceutically acceptable carrier.

In one or more embodiments of any of the above aspects, the pain iscaused by inflammation. In some embodiments, the pain is caused by theinitiation of the inflammatory response. In some embodiments, the painis associated with hyperalgesia.

In one or more embodiments, Compound 1 does not cross the blood-brainbarrier. In one or more embodiments, Compound 1 does not affect thecentral nervous system. In one or more embodiments, Compound 1 activateskappa opioid receptors. In one or more embodiments, Compound 1 activatesde/ta opioid receptors. In one or more embodiments, Compound 1 activateskappa and delta opioid receptors. In one or more embodiments, Compound 1does not significantly activate mu receptors.

In one or more embodiments, the pain is chronic pain or subacute pain.In one or more embodiments, the chronic pain is arthritis pain, low backpain, neuropathic pain, visceral pain, pain due to cancer, pain due toinjury, pain due to joint inflammation, pain due to back disorders, orneck pain. In one or more embodiments, the pain due to cancer is causedby cancer involving intraperitoneal abdominal and pelvic organs or bonecancer. In one or more embodiments, the pain due to injury is caused bybone, ligament, or tendon injury. In some embodiments, the pain is dueto irritable bowel syndrome or interstitial cystitis. In someembodiments, the pain is due to inflammatory arthritis.

In one or more embodiments, Compound 1 reduces pain to a similar orgreater degree as a central-nervous system-acting opioid. In one or moreembodiments, the central-nervous system-acting opioid activates a mureceptor. In one or more embodiments, the central-nervous system-actingopioid is morphine.

In one or more embodiments, administrating Compound 1 does not result inany central-nervous system side effects. In one or more embodiments, thecentral nervous system side-effects are addiction, sedation, impairedmentation, somnolence, respiratory depression, nausea, constipation,dysphoria, or seizures. In one or more embodiments, administratingCompound 1 does not result in addiction.

In one or more embodiments, Compound 1 results in synergistic activationof kappa and de/ta opioid receptors. In one or more embodiments, thesynergy results from the delta effect enhancing the kappa effect. In oneor more embodiments, administration of Compound 1 is similar to orsuperior to a kappa receptor agonist for treatment of pain (e.g.,inflammatory pain). In one or more embodiments, administration ofCompound 1 is similar to or superior to a kappa receptor agonist fortreatment of hyperalgesia. In one or more embodiments, administration ofCompound 1 results in reduced urinary output compared to a kappareceptor agonist.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graphic depicting the baseline biochemical state ofuninflamed tissue.

FIG. 1B is a graphic depicting the biochemical response in tissue toinflammation.

FIG. 2A is a line graph of the pain behaviors exhibited by the mice thatreceived each dose of vehicle or drug as set forth in Example 1.

FIG. 2B is a bar graph of the pain behaviors exhibited by the mice thatreceived each dose of vehicle or drug as set forth in Example 1.

FIG. 3 is a bar graph depicting the urine output of mice that receivedeach dose of Compound 1 or IC1204448 as set forth in Example 1.

FIG. 4 is a bar graph comparing the joint compression thresholds betweenthe ipsilateral and contralateral legs of injured rats at various timepoints in Example 2.

FIG. 5 is a bar graph showing the effects of Compound 1 on CFA-inducedmechanical hyperalgesia in Example 2.

FIG. 6 is a bar graph comparing the paw compression thresholds betweenthe ipsilateral and contralateral legs of injured rats at various timepoints in Example 3.

FIG. 7 is a bar graph showing the effect of Compound t on SNL-inducedmechanical hyperalgesia in Example 3.

FIG. 8 is a bar graph depicting the percent of weight bearing of theinjured leg for untreated rats after the development of bone cancer atvarious time points in Example 4.

FIG. 9 is a bar graph showing the effect of Compound 1 on bone cancerpain as measured by the percent of weight bearing by the injured leg.

DETAILED DESCRIPTION OF THE DISCLOSURE

Opioid analgesics are among the most important and powerful analgesicsavailable. Many existing preparations rely on the mu opioid receptor inthe central nervous system (i.e., brain and spinal cord) for theiractivity. Unfortunately, mu opioid agonism in the central nervous systemcan be responsible for some of the serious adverse effects associatedwith opioid analgesia including life-threatening respiratory depressionand addiction. Further, mu opioid agonism can be responsible for othertroublesome side effects including impaired mentation, somnolence,nausea and constipation.

In the peripheral tissues, mu receptors can be much less involved in thepain pathway. Different types of opioid receptors, namely the kappa anddelta opioid receptors, can often be present in peripheral sensorynerves as well as the central nervous system. These too can beassociated with other unwanted adverse effects, including dysphoria andseizures, due to their activity in the central nervous system.

The restriction of mu opioid agents to the peripheral nervous system(i.e., keeping opioid agents out of the central nervous system to avoidinteraction with mut receptors there), can help avoid central adverseeffects including the addictive potential and respiratory depression.However, because mu opioid receptors do not play a major role in theperipheral pain pathway, the effect of mu opioid agonism in theperiphery can have minimal impact on analgesia.

Peripherally-restricted kappa opioid agonists (e.g., ICI204448) cansometimes provide relatively modest analgesia. The kappa receptor is, toa significant extent, under the influence of the normally quiescentdelta opioid receptor through heterodimerization of the kappa and deltareceptors. However, in the presence of inflammation, the delta receptoris unsequestered, allowing it to not only participate in analgesiaitself, but also to boost the activity of the kappa receptor throughallosteric modulation. Without wishing to be bound by theory, in thepresence of inflammation, having both kappa and delta activity in theperiphery can enhance the analgesic effect.

The present disclosure teaches a dual-acting, peripherally-restrictedopioid with both kappa and delta effect, but minimal mu effect (i.e.,Compound 1, below). In some embodiments, Compound 1 has significantlyimproved analgesia compared to other analgesics such as pure kappaagonist agents (e.g., ICI204448) and/or pure mu agonist agents (e.g.,morphine or heroin). In some embodiments, Compound 1 has increasedanalgesic effect in the presence of inflammation. In some embodiments,Compound 1 has limited potential for addiction (e.g., no potential foraddiction). In some embodiments Compound 1 has limited potential for(e.g., no potential for) somnolence, respiratory depression, seizure,dysphoria, or constipation.

Throughout this disclosure, various patents, patent applications andpublications are referenced. The disclosures of these patents, patentapplications and publications in their entireties are incorporated intothis disclosure by reference in order to more fully describe the stateof the art as known to those skilled therein as of the date of thisdisclosure. This disclosure will govern in the instance that there isany inconsistency between the patents, patent applications andpublications and this disclosure.

Definitions

For convenience, certain terms employed in the specification, examplesand claims are collected here. Unless defined otherwise, all technicaland scientific terms used in this disclosure have the same meanings ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs. The initial definition provided for a group or termprovided in this disclosure applies to that group or term throughout thepresent disclosure individually or as part of another group, unlessotherwise indicated.

“Inflammatory Response” (or inflammatory cascade) refers to the innateimmune response to injury involving the elaboration of chemokines andinflammatory peptides such as bradykinin

“VGCC” refers to voltage-gated calcium channel.

“B2R refers to bradykinin receptor B2, which is constitutively presentin normal tissues.

“DPDPE” refers to (D-Pen2,D-Pen5)-Enkephalin, a selective delta opioidagonist.

“hyperalgesia” refers to an increased sensitivity to painful stimuli.

As used herein, “DOR” refers to delta opioid receptor. The term “KOR”refers to kappa opioid receptor, and “MOR” refers to mu opioid receptor.

As used herein, “GRK2” refers to G protein-coupled receptor kinase-2.

As used herein, “BK” refers to bradykinin.

As used herein, “PKC” refers to protein kinase C.

As used herein, “RKIP” refers to Raf kinase inhibitory protein.

As used herein, “CFA” refers to Complete Freund's Adjuvant.

As used herein, “JCT” refers to joint compression threshold.

As used herein, “SEM” refers to standard error of the mean

As used herein, “IP” refers to intraperitoneal administration.

As used herein, “PO” refers to “per os” or administration by mouth.

As used herein, “ANOVA” refers to analysis of variance.

As used herein, “IACUC” refers to the Institutional Animal Care and UseCommittee.

As used herein, “SNL” refers to spinal nerve ligation.

“Pharmaceutically acceptable carrier” includes without limitation anyadjuvant, carrier, excipient, glidant, sweetening agent, diluent,preservative, dye/colorant, flavor enhancer, surfactant, wetting agent,dispersing agent, suspending agent, stabilizer, isotonic agent, solvent,or emulsifier which has been approved by the United States Food and DrugAdministration as being acceptable for use in humans or domesticanimals.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

A “pharmaceutical composition” refers to a formulation of a compound ofthe invention and a medium generally accepted in the art for thedelivery of the biologically active compound to mammals, e.g., humansSuch a medium includes all pharmaceutically acceptable carriers,diluents or excipients therefor.

Subjects or patients “in need of treatment” with a compound of thepresent disclosure include patients with diseases and/or conditions thatcan be treated with the compounds of the present disclosure to achieve abeneficial therapeutic result. A beneficial outcome includes anobjective response or a subjective response including self-reportedreduction in pain. For example, a patient in need of treatment issuffering from pain and/or hyperalgesia. In some cases the patient issuffering from subacute (e.g., chronic) pain that can be caused by, forinstance, arthritis or other inflammation.

As used herein, an “effective amount” (or “therapeutically effectiveamount”) of a compound disclosed herein, is a quantity that results in abeneficial clinical outcome (e.g., pain reduction) of the conditionbeing treated with the compound compared with the absence of treatment.The amount of the compound or compounds administered will depend on thedegree, severity, and type of the disease or condition, the amount oftherapy desired, and the release characteristics of the pharmaceuticalformulation. It will also depend on the subject's health, size, weight,age, sex and tolerance to drugs. Typically, the compound is administeredfor a sufficient period of time to achieve the desired therapeuticeffect.

The terms “treatment,” “treat,” and “treating,” are meant to include thefull spectrum of intervention in patients with “pain” with the intentionto reduce, mollify or eliminate the pain from which the patient issuffering. Treating can be curing, improving, or at least partiallyameliorating the patient's condition (e.g., pain).

“Prevention” or “Preemption” include reducing the expected oranticipated symptoms of a disease or condition before they are exhibitedby a subject. For example, as set forth herein, pain (e.g., inflammatorypain) can be prevented or preempted in a subject at risk for pain (e.g.,a subject with an inflammatory condition) by treatment with Compound 1.In some embodiments, treatment with Compound 1 in subjects who do notyet have pain can prevent the subject from experiencing pain. Forexample, as used herein, the onset of pain can be prevented by treatinga subject with Compound 1 before the subject undergoes an event that maycause pain (e.g., an operation). For example, as used herein, aworsening of pain (e.g., more intense pain as self-reported by thesubject) can be prevented by treating a subject with Compound 1 beforethe subject undergoes an event that may cause pain (e.g., an operation).

“Cancer” as defined herein refers to a new growth which has the abilityto invade surrounding tissues, metastasize (spread to other organs) andwhich may eventually lead to the patient's death if untreated. “Cancer”can be a solid tumor or a liquid tumor.

Compound 1

As used herein, Compound 1 is understood as4-(((2S,5S,8S)-2-isobutyl-8-isopropyl-1-phenethyl-2,3,5,6,8,9-hexahydro-1H-diimidazo[1,2-d:2′,1′-g][1,4]diazepin-5-yl)methyl)phenol.Compound 1 is a peripherally restricted opioid with agonist activityagainst kappa and delta receptors and the structure of Compound 1 isgiven below:

ICI204448 is a peripherally-restricted selective kappa opioid agonist.Its effects are evaluated in Example 1, below. It has the structurebelow:

Celecoxib is a COX-2 selective nonsteroidal anti-inflammatory drug ofthe formula:

Gabapentin is a drug used to treat neuropathic pain. It has thestructure:

Proposed Mechanism of Action of Compound 1

FIG. 1 sets forth a proposed mechanism of action for Compound 1. Withoutwishing to be bound by theory, FIG. 1A shows a proposed native state fornon-inflamed tissue. As shown in FIG. 1A, G protein-coupled receptorkinase-2 (GRK2) can bind to a delta opioid receptor (DOR), inactivatingthe DOR. Thus, the DOR does not affect the sensation of pain in thenon-inflamed state.

FIG. 1B shows a proposed state of inflamed tissue. Without wishing to bebound by theory, in the inflamed state, proinflammatory bradykinin (BK)can stimulate GRK2 movement away from DOR and onto Raf kinase inhibitoryprotein (RKIP). This chain of events can allow the activation of theDOR. In particular, protein kinase C (PKC)-dependent RKIPphosphorylation associated with the binding of BK can induce GRK2sequestration, restoring functionality of DOR in sensory neurons. ActiveDOR can then be available to participate in reducing the sensation ofpain in subjects, e.g., in the inflamed state (Brackley et al., CellRep. 2016, 16(10):2 686-2698).

Furthermore, active DOR can allosterically enhance the activity of kappaopioid receptors (KOR), which are constitutively present in peripheralsensory neurons and are available to synergistically participate inreducing the sensation of pain in subjects (e.g., in the inflamedstate). Accordingly, in some embodiments, GRK2 sequestration, e.g., uponinflammatory stimulus, can make DORs and KORs more efficient, providingthe opportunity to reduce the sensation of pain in inflamed subjects.

For example, without wishing to be bound by theory, DORs and KORs canform heterodimers in peripheral sensory neurons (i.e., DOR-KORheterodimers). Without wishing to be bound by theory, allostericineractions in DOR-KOR heterodimers can modulate sensitivity to painfulstimuli in the presence of inflammation. The activity of theseheterodimers in animal models of pain has been demonstrated inperipheral sensory neurons (Berg et al., Mol Pharmacol. 2012; 81(2):264-72). Allosteric interaction between the kappa and delta componentsis thought to contribute to the enhancement of kappa-mediated analgesiaby delta agonists. Evidence for DOR-KOR heteromers in peripheral sensoryneurons includes coimmunoprecipitation of DOR with KOR; that a DOR-KORheteromer selective antibody augmented the antinociceptive effect ofDPDPE (delta agonist) in vivo; and the DOR-KOR heteromer agonist 6-GNTIinhibited adenylyl cyclase activity in vitro as well as PGE2-stimulatedthermal allodynia in vivo. Accordingly, without wishing to be bound bytheory, DOR-KOR heteromers can exist in primary sensory neurons and KORactive agents can act as modulators of DOR agonist responses, forinstance through allosteric interactions between the promoters of theDOR-KOR heteromer.

Without wishing to be bound by theory, because Compound 1 does not crossthe blood-brain barrier (BBB), it is proposed that Compound 1 does notsuffer from the same shortcomings as traditional opioids. Specifically,because Compound 1 does not cross the BBB, and therefore does notsignificantly interact with mu opioid receptors, Compound 1 is lessprone to result in addiction and other CNS-associated side effects suchas constipation, impaired mentation, somnolence, and the like. Thus, insome embodiments, Compound 1 does not suffer from the same drawbacks astraditional opioids. In some embodiments, Compound 1 does not result incentral nervous system side effects such as addiction.

Additionally, without wishing to be bound by theory, Compound 1 iseffective at treating pain, e.g., inflammatory and/or chronic pain. Asset forth above, Compound 1 is an effective agonist at both the kappaand delta opioid receptors. This dual activity can result in high levelsof pain relief for patients, without the deleterious central nervoussystem effects associated with traditional opioids. Accordingly, in someembodiments, Compound 1 is administered to patients with greater safetythan traditional opioid analgesics.

Formalin Model of Pain in Rodents

Without wishing to be bound by theory, as set forth in Example 1, micewere treated with formalin in a standard model for pain assessment inmice. Without wishing to be bound by theory, the formalin test in miceevaluates pain in two phases. Phase 1 can last for about 5-10 minutesafter injection into the hind paws of mice. Phase 1 can evaluate themice's response to the acute pain immediately following formalin (anirritating substance) injection. Accordingly, in some embodiments phase1 of the formalin model can evaluate pain caused by the stimulation ofnociceptors (i.e., phase 1 can evaluate nociceptive or acute pain).

Without wishing to be bound by theory, phase 2 of the formalin model canbegin about twenty minutes after the initial injection of formalin.Phase 2 can represent and evaluate hyperalgesia initiated by theinflammatory process. For example, the inflammatory response can betriggered by tissue damage with subsequent sensitization of nociceptors.This sensitization process can take about twenty minutes to develop andcan then be sustained for about 60 minutes or longer after injection.Accordingly, phase 2 measures inflammatory pain and the responsethereto.

As set forth in Example 1 below, mice were tested in a formalin modelfor pain and were subsequently treated with (i) inert vehicle; (ii) lowdose of a peripherally-restricted kappa opioid agonist (i.e.,ICI204448), (iii) high dose of a peripherally-restricted kappa opioidagonist (i.e., ICI204448); (iv) low dose of Compound 1; and (v) highdose of Compound 1. Compared with inert vehicle and ICI204448, Compound1 reduced pain in mice treated with formalin at about 20-25 minutes,25-30 minutes, and 30-35 minutes (i.e., during phase 2).

As set forth in Example 1 and as shown in FIG. 2, only a small dose ofCompound 1 was needed to produce the same effect as a high dose of theperipherally-restricted kappa opioid receptor agonist (i.e., ICI204448)in phase 2 of the formalin model. Moreover, the high dose of Compound 1was shown to produce complete elimination of the phase 2 hyperalgesicpain response in mice.

Accordingly, in some embodiments, the present disclosure teaches thetreatment of pain (e.g., pain caused by inflammation or the initiationof the inflammatory response) by administering to a subject in needthereof an effective amount of Compound 1. In some embodiments, themagnitude of the reduction in pain is substantially similar to thereduction in pain caused by a central-nervous system-acting opioid(e.g., morphine). In some embodiments, Compound 1 is effective atreducing hyperalgesia (e.g., more effective than a kappa opioid receptoralone). In some embodiments, Compound 1 can simultaneously activatekappa and delta opioid receptors to result in a synergistic reduction inpain at lower doses than is observed with other drugs such as pure kappaagonists (e.g., ICI204448). For instance, it was found that Compound 1was at least as effective as ICI204448 in reducing time spent on pain(i.e., nociceptive) behaviors at 30-35 minutes even though Compound 1was administered at a dose of less than 10% of the amount of ICI204448on a molar basis (FIGS. 2A and 2B).

As set forth in Example 1 and FIG. 3, mice that were treated withCompound 1 were found to produce less urine than mice treated with thekappa agonist IC1204448 over a 6-hour collection period followingformalin testing. Accordingly, in some embodiments, treatment withCompound 1 can be less likely to result in diuresis compared with otherperipherally-restricted kappa opioid agonists (e.g., ICI204448).

Furthermore, as set forth in Example 1, animals were treated withCompound 1 before formalin injection. As shown in FIGS. 2A and 2B,animals that were treated with a high dose of Compound 1 did not exhibitany substantial pain behaviors at 20-25 min, 25-30 min, or 30-35 min.Accordingly, Example 1 suggests that Compound 1 can be administeredbefore an injury (e.g., an injury that is likely to cause inflammation)and prevent the sensation of pain (e.g., pain due to inflammation).Accordingly, in some embodiments Compound 1 can be used to prevent pain(e.g., pain due to inflammation).

Arthritis Model of Pain in Rats

Example 2 below evaluated the efficacy of a single intraperitoneal (IP)injection of Compound 1 in a model of rheumatoid arthritis in rats. Asdemonstrated in Example 2, a single intraperitoneal dose of Compound 1significantly reduced established mechanical hyperalgesia due toCFA-induced rheumatoid arthritis in the rat in a time- anddose-dependent manner.

Without wishing to be bound by theory, as set forth in Example 2, ratswere administered Compete Freund's Adjuvant (CFA) to produce anarthritis-like response. After two weeks, and once an inflammatoryresponse had developed, rats were treated with inert vehicle, Compound1, or celecoxib (a cyclo-oxygenase-2 (COX-2) anti-inflammatory drug).Joint compression thresholds (JCTs) were measured before and aftertreatment with vehicle, Compound 1 or celecoxib as a proxy for painthresholds.

FIG. 4 shows the contrast between the JCTs for the injured (i.e.,ipsilateral) vs non-injured (i.e., contralateral) legs for ratsadministered vehicle FIG. 4 demonstrates that for rats that did notreceive either Compound 1 or celecoxib, the JCT for the injured leg wasabout two-thirds that of the JCT for the non-injured leg, suggestingthat the injured leg was more painful than the non-injured leg.

FIG. 5 compares the JCTs at one, two and four hours after administrationof vehicle, Compound 1, or celecoxib. As shown in FIG. 5, all threedoses of Compound 1 (i.e., 1, 5 and 10 mg/kg) led to significantincreases in JCT, suggesting a decrease in pain sensation in rats. Theresults demonstrate that Compound 1 was able to reverse mechanicalhyperalgesia in the injured leg after administration.

Accordingly, Example 2 is an exemplary model of a chronic pain statewith a predominant inflammatory component. The combination ofperipherally restricted delta and peripherally restricted kappa agonismfrom Compound 1 resulted in an attenuation of pain behaviors that wasgreater than that seen with an anti-inflammatory drug. Specifically, theresponse in the rheumatoid arthritis model was comparable or superior tocelecoxib.

Neuropathic Pain Model in Rats

Example 3 below evaluated the efficacy of a single intraperitonealinjection of Compound 1 and the comparator, gabapentin, in the spinalnerve ligation (SNL) model for neuropathic pain in the rat. Asdemonstrated in Example 3, intraperitoneal injection of Compound 1produced a time- and dose-dependent analgesic effect on mechanicalhyperalgesia associated with SNL-induced neuropathic pain in the rat.

Without wishing to be bound by theory, as set forth in Example 3, ratswere subject to spinal nerve ligation to produce a neuropathic-typeresponse. After fifteen days, once a neuropathic response had developed,rats were treated with inert vehicle, Compound 1, or gabapentin. Pawcompression thresholds were measured before and after treatment withvehicle, Compound 1 or gabapentin as a proxy for pain thresholds. Pawcompression thresholds were measured using the same technique and deviceas the joint compression thresholds outlined above in the arthritis painmodel, but were evaluated on the paw instead of on the ankle.

FIG. 6 shows the contrast between paw compression thresholds for theinjured (i.e., ipsilateral) vs non-injured (i.e., contralateral) legsfor rats administered vehicle. FIG. 4 demonstrates that for rats thatdid not receive either Compound 1 or gabapentin, the paw compressionthresholds for the injured leg was about one half that of the pawcompression threshold of the non-injured leg, suggesting that theinjured leg is more painful than the non-injured leg.

FIG. 7 compares the paw compression thresholds at one, two, and fourhours after administration of vehicle, Compound 1, or gabapentin. Asshown in FIG. 7, the 5- and 10-mg doses of Compound 1 led to significantincreases in paw compression thresholds at the 2- and 4-hour timepoints, suggesting a decrease in pain sensation in the injured leg.

Accordingly, Example 3 suggests that in chronic neuropathic pain states,characterized by relative mu-opioid resistance and significantinflammatory response, moderate doses of Compound 1 are as effective orsuperior to gabapentin. Thus, in some embodiments the present disclosureprovides for the treatment of neuropathic (e.g., chronic neuropathic)pain comprising administering Compound 1.

Bone Cancer Model of Pain in Rats

Without wishing to be bound by theory, Example 4 below evaluated theefficacy of a single intraperitoneal injection of Compound 1, and thecomparator, subcutaneous morphine, in the MRMT-1 model of osteolyticcancer pain in rats. As shown in Example 4, Compound 1 administered at10 mg/kg (IP) had a significant effect on osteolytic bone cancer paininduced by MRMT-1 inoculation with a slower onset compared to morphine.

As set forth in Example 4, rats were injected with MRMT-1 cancer cellsto induce bone cancer in one of the hind legs. After 21 days, after thedevelopment of bone cancer, the rats were evaluated to measure thepercent of weight bearing of each hind leg (i.e., injured vs.non-injured) as a proxy for pain in each leg.

FIG. 8 shows the percent weight bearing scores for rats that wereadministered vehicle at time points pre-injury, pre-injection withvehicle, and at 1, 2 and 4 hours after injection with vehicle FIG. 8shows that there was substantial variability between the pre-dosebaseline measurement and the 1- and 2-hour time points, partially due tothe fact that not all rats exhibited symptoms bone cancer. As a resultof this variability, and in the interest of obtaining a reliable dataset, the originally-proposed grouping of five groups with ten rats eachwas reconsidered in favor of three groups with thirteen rats each. Thethree evaluated groups were: Group 1 (treated with vehicle); Group 4(treated with 10 mg/kg Compound 1), and Group 5 (treated with 6 mg/kgmorphine). Groups 2 and 3, which had originally been proposed toevaluate the effects of Compound 1 at 1 and 5 mg/kg respectively, werenot evaluated.

FIG. 9 shows the percent weight bearing score for rats at one, two andfour hours after administration with vehicle, Compound 1, or morphine.As shown in FIG. 9, Compound 1 had a significant effect on osteolyticbone cancer pain induced by MRMT-1 cancer cells with a slower onsetcompared to morphine.

Bone cancer pain, despite relative resistance to opioids, typically onlyresponds to strong mut-opioid treatment. The degree of inflammation,while present, is not as pronounced as in the previously referencedchronic pain conditions. Compound 1 produced a delayed reduction in painbehaviors associated with bone cancer pain in a single dose study.Without wishing to be bound by theory, chronic dosing could be useful toaddress chronic pain (e.g., pain due to cancer such as bone cancer).

Pain Indications

Thus, in some embodiments, Compound 1 can be used in the treatment ofpain. The pain can be inflammatory pain, or pain caused by theinitiation of the inflammatory response in a subject. In someembodiments, the pain can be due to an autoimmune disorder or otherinflammatory disorder. In some embodiments, the pain can be due toarthritis. For example, the pain can be due to rheumatoid arthritis,osteoarthritis (e.g., osteoarthritis with synovitis) posttraumaticarthritis, or inflammatory arthritis.

In some embodiments, the pain is due to inflammatory bowel disease,irritable bowel syndrome, peritonitis, pleuritic pain, pelvicinflammation, fibromyalgia, or interstitial cystitis.

In some embodiments, the pain is neuropathic pain. For example, the paincan be due to complex regional pain syndrome, radiculitis, orinflammatory neuritis. In some embodiments, the pain is due to neuralgia(e.g., postherpetic neuralgia).

In some embodiments, the pain can be due to cancer. The cancer can beprimary cancer or metastatic cancer. In some embodiments, the pain isdue to cancer involving the thoracic organs, intraperitoneal organs,abdominal organs, pelvic organs, or bone cancer. Pain can be due tocarcinomatosis. Pain can be due to an infectious process of theintrapleural space and/or intrapleural inflammation (e.g., pleurisy).Pain can be due to intraperitoneal inflammatory processes. For example,pain can be due to intraperitoneal inflammatory processes involving thepancreas (e.g., pancreatitis), liver, bowel, spleen, or urinary bladder(e.g., pelvic inflammatory disease and/or interstitial cystitis).

In some embodiments, the pain can be due to injury (e.g., tissueinjury). In some embodiments, the pain is due to joint injury, bursainjury, muscle injury, bone injury, ligament injury, or tendon injury.

In some embodiments, the pain is arthritis pain, low back pain (e.g.,pain due to back disorders), neuropathic pain, visceral pain, or neckpain. In some embodiments, the back (e.g., low back) and/or neck paincan be with or without radiculopathy Pain can be due to musculoskeletalinjury, tendonitis, and/or myofascial pain syndrome. The pain can bechronic pain or subacute pain.

In some embodiments, the pain is due to chronic inflammatory pain states(e.g., chronic inflammatory pain states with hyperalgesia). In someembodiments, the pain is due to acute and/or subacute pain states (e.g.,acute and/or subacute pain states with hyperalgesia). For example, insome embodiments, the pain can be due to postoperative and/orposttraumatic pain (e.g., burn pain).

Pharmaceutical Compositions and Methods of Treatment

The present disclosure is also directed to methods of treatmentinvolving the administration of Compound 1 of the present disclosure, ora pharmaceutical composition comprising Compound 1. The pharmaceuticalcomposition or preparation described herein may be used in accordancewith the present disclosure, e.g., for the treatment of pain (e.g.,inflammatory pain) or hyperalgesia.

Compound 1, utilized in the treatment methods of the present disclosure,as well as the pharmaceutical compositions comprising it, mayaccordingly be administered alone, or as part of a treatment protocol orregiment that includes the administration or use of other beneficialcompounds (e.g., as part of a combination therapy).

In using the pharmaceutical compositions of Compound 1 described herein,pharmaceutically acceptable carriers can be either solid or liquid.Solid forms include powders, tablets, dispersible granules, capsules,cachets and suppositories. The powders and tablets can comprise fromabout 5 to about 95 percent active ingredient (i.e., Compound 1).Suitable solid carriers are known in the art, e.g., magnesium carbonate,magnesium stearate, talc, sugar or lactose. Tablets, powders, cachetsand capsules can be used as solid dosage forms suitable for oraladministration. Examples of pharmaceutically acceptable carriers andmethods of manufacture for various compositions may be found in A.Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition,(1990), Mack Publishing Co., Easton, Pa., which is hereby incorporatedby reference in its entirety.

Liquid form preparations include solutions, suspensions and emulsions.For example, water or water-propylene glycol solutions for parenteralinjection or addition of sweeteners and opacifiers for oral solutions,suspensions and emulsions. Liquid form preparations may also includesolutions for intranasal administration.

Liquid, particularly injectable, compositions can, for example, beprepared by dissolution, dispersion, etc. For example, the disclosedcompound is dissolved in or mixed with a pharmaceutically acceptablesolvent such as, for example, water, saline, aqueous dextrose, glycerol,ethanol, and the like, to thereby form an injectable isotonic solutionor suspension. Proteins such as albumin, chylomicron particles, or serumproteins can be used to solubilize the disclosed compounds.

Parenteral injectable administration is generally used for subcutaneous,intramuscular or intravenous injections and infusions. Injectables canbe prepared in conventional forms, either as liquid solutions orsuspensions or solid forms suitable for dissolving in liquid prior toinjection. Aerosol preparations suitable for inhalation may also beused. These preparations may include solutions and solids in powderform, which may be in combination with a pharmaceutically acceptablecarrier, such as an inert compressed gas, e.g., nitrogen. Alsocontemplated for use are solid form preparations that are intended to beconverted, shortly before use, to liquid form preparations for eitheroral or parenteral administration. Such liquid forms include solutions,suspensions and emulsions.

Dosage

The amount and frequency of administration of Compound 1 and/or thepharmaceutically acceptable salts thereof will be regulated according tothe judgment of the attending clinician considering such factors as age,condition and size of the patient as well as severity of the symptomsbeing treated. Effective dosage amounts of Compound 1, when used for theindicated effects, range from about 0.5 mg to about 5000 mg of Compound1 as needed to treat the condition. Compositions for in vivo or in vitrouse can contain about 0.5, 5, 20, 50, 75, 100, 150, 250, 500, 750, 1000,1250, 2500, 3500, or 5000 mg of Compound 1, or, in a range of from oneamount to another amount in the list of doses. A typical recommendeddaily dosage regimen for oral administration can range from about 1mg/day to about 500 mg/day or 1 mg/day to 200 mg/day, in a single dose,or in two to four divided doses. In one embodiment, the daily doseregimen is 150 mg.

In some embodiments, Compound 1 can be administered for one day, twodays, three days, four days, five days, six days, or seven days. In someembodiments, Compound 1 can be administered one week, two weeks, threeweeks, or four weeks. In some embodiments, Compound 1 can beadministered one month, two months, three months, four months, fivemonths, six months, or longer. In some embodiments, Compound 1 can beadministered indefinitely (e.g., chronic dosing).

Compound 1, with or without an additional therapeutic agent, can beadministered by any suitable route. The compound can be administratedorally (e.g., dietary) in capsules, suspensions, tablets, pills,dragees, liquids, gels, syrups, slurries, and the like. Methods forencapsulating compositions (such as in a coating of hard gelatin orcyclodextran) are known in the art (Baker, et al., “Controlled Releaseof Biological Active Agents”, John Wiley and Sons, 1986, which is herebyincorporated by reference in its entirety). Compound 1 can beadministered to the subject in conjunction with an acceptablepharmaceutical carrier as part of a pharmaceutical composition. Theformulation of the pharmaceutical composition will vary according to theroute of administration selected. Suitable pharmaceutical carriers maycontain inert ingredients which do not interact with the compound. Thecarriers can be biocompatible, i.e., non-toxic, non-inflammatory,non-immunogenic and devoid of other undesired reactions at theadministration site. Additionally, Compound 1 can be administeredparenterally, subcutaneously, intramuscularly or intravenously. Compound1 can be administered intraperitoneally.

Illustrative pharmaceutical compositions are tablets and gelatincapsules comprising Compound 1 and a pharmaceutically acceptablecarrier, such as a) a diluent, e.g., purified water, triglyceride oils,such as hydrogenated or partially hydrogenated vegetable oil, ormixtures thereof, corn oil, olive oil, sunflower oil, safflower oil,fish oils, such as EPA or DH A, or their esters or triglycerides ormixtures thereof, omega-3 fatty acids or derivatives thereof, lactose,dextrose, sucrose, mannitol, sorbitol, cellulose, sodium, saccharin,glucose and/or glycine; b) a lubricant, e.g., silica, talcum, stearicacid, its magnesium or calcium salt, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chlorideand/or polyethylene glycol; for tablets also; c) a binder, e.g.,magnesium aluminum silicate, starch paste, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose, magnesium carbonate,natural sugars such as glucose or beta-lactose, corn sweeteners, naturaland synthetic gums such as acacia, tragacanth or sodium alginate, waxesand/or polyvinylpyrrolidone, if desired; d) a disintegrant, e.g.,starches, agar, methyl cellulose, bentonite, xanthan gum, algic acid orits sodium salt, or effervescent mixtures; e) absorbent, colorant,flavorant and sweetener, f) an emulsifier or dispersing agent, such asTween 80, Labrasol, HPMC, DOSS, caproyl 909, labrafac, labrafil, peceol,transcutol, capmul MCM, capmul PG-12, captex 355, gelucire, vitamin ETGPS or other acceptable emulsifier; and/or g) an agent that enhancesabsorption of the compound such as cyclodextrin,hydroxypropyl-cyclodextrin, PEG400, PEG200.

If formulated as a fixed dose, such pharmaceutical compositions employCompound 1 within the dosage range described herein, or as known tothose skilled in the art.

Since Compound 1 is intended for use in pharmaceutical compositions askilled artisan will understand that it can be provided in substantiallypure form for example, at least 60% pure, at least 75% pure, at least85% pure, at least 98% pure and at least 99% pure (w/w). Thepharmaceutical preparation may be in a unit dosage form. In such form,the preparation is subdivided into suitably sized unit doses containingappropriate quantities of Compound 1, e.g., an effective amount toachieve the desired purpose as described herein (e.g., pain reduction).

EXAMPLES

The disclosure is further illustrated by the following examples, whichare not to be construed as limiting this disclosure in scope or spiritto the specific procedures herein described. It is to be understood thatthe examples are provided to illustrate certain embodiments and that nolimitation to the scope of the disclosure is intended thereby. It is tobe further understood that resort may be had to various otherembodiments, modifications, and equivalents thereof which may suggestthemselves to those skilled in the art without departing from the spiritof the present disclosure and/or scope of the appended claims.

Example 1—Formalin Model of Pain in Mice

The objective of this study was to evaluate the effects of Compound 1and a peripherally-restricted kappa agonist (IC1204448) onformalin-evoked spontaneous nociceptive behaviors in mice. The study wasperformed by video recording of formalin-induced nociceptive behaviorand then off-line scoring using a computer.

Subcutaneous plantar injection of formalin causes a bi-phasicnocifensive behavioral response in rodents. The early phase (phase 1)lasts for about 5-10 minutes, following which an interphase occurswithout any discernible nociceptive reactions, after which the latephase (phase 2) nociceptive reaction ensues continuing from about 20-60min following formalin injection. Thus, phase 2 of the formalin model isa model of continuously present, persistent pain, and is widely used forrapid screening of novel analgesic compounds. The model encompassesinflammatory, neurogenic and central mechanisms of nociception, and thelate phase (phase 2), in particular, is considered as a pharmacodynamicsurrogate of central sensitization.

In the present study, the effects of Compound 1 and IC1204448 wereassessed from 0-5 minutes for the early phase (phase 1) and from 20-35minutes for the late phase (phase 2) of formalin-induced nociceptivebehavior.

Methods

Following IACUC approval and acclimation C57BL6 mice (Charles RiverCanada Inc.), 20-30 g, were randomly assigned into groups with 8 miceper group as provided in Table 1.

TABLE 1 Study Design Dose Dose Pre- Group level volume treatment Groupstreatment (mg/kg) Route (mL/kg) time N 1 Vehicle 0 IP 20 30 min 8 (1:1:8ethanol:Tween 80:0.9% saline) 2 ICI204448 - 1 IP 20 30 min 8 Low Dose 3ICI204448 - 10 IP 20 30 min 8 High Dose 4 Compound 1 - 1 IP 20 30 min 8Low Dose 5 Compound 1 - 10 IP 20 30 min 8 High Dose

All animals were acclimated to the observation chamber for about 15minutes immediately prior to formalin injection. All animals received a30 μL injection of freshly prepared formalin solution (5% in phosphatebuffered saline; PBS) intra-plantarly (i.pl.) into the left hind paw.Animals were administered the vehicle, IC1204448 or Compound 1intraperitoneally (IP) 30 minutes before formalin injection as depictedin the Table 1 above.

Following injection of the formalin all animals were returnedimmediately to the observation chamber and formalin-evoked spontaneousnociceptive behaviors in the mice were continuously recorded for 0-40minutes using a commercial camcorder. The camera was turned on at least5 minutes before formalin injection and verified for proper functioning.

Scoring from the recorded video files were done off-line using acomputer by a blinded observer who has been validated to score suchnociceptive behaviors in rodents. The total (cumulative) time spent in a5-minute bin was recorded using a stop-watch for the followingnociceptive behaviors: biting and licking of the formalin-injected paw.

Effects of the ICI204448 or Compound 1 were assessed in the followingtime periods. 0-5 minutes for the early phase (phase 1) and 20-35minutes for the late phase (phase 2).

Mice were injected with formalin in the hind paw after pretreatment withvehicle, high and low doses of a peripherally-restricted kappa opioidagonist (ICI204448) and high and low doses of theperipherally-restricted Compound 1. Pain was measured for 35 minutesafter injection. Additionally, urine output was measured using metaboliccages in each group of mice. Total urine volumes were collected over sixhours

Results

FIGS. 2A and 2B show a line graph and a bar graph, respectively, of thepain behaviors exhibited by the mice that received each dose of vehicleor drug. As shown in FIGS. 2A and 2B, compared with inert vehicle andIC1204448. Compound 1 reduced pain behaviors in mice treated withformalin at about 20-25 minutes, 25-30 minutes, and 30-35 minutes (i.e.,phase 2). Additionally, only a small dose of Compound 1 (1 mg/kg) wasneeded to produce the same effect as a high dose of IC1204448. Moreover,the high dose of Compound 1 (10 mg/kg) was shown to produce completeelimination of the pain response in mice. A comparison of p values as afunction of dose is given below in Table 2.

TABLE 2 Comparison of p Values by Dose Comparison Timeframe p ValueCompound 1 (high dose) v. vehicle 30-35 min p < 0.03 Compound 1 (highdose) v. vehicle 20-35 min p < 0.003 Compound 1 (high dose) v. KORagonist (high dose) 20-35 min p < 0.01 KOR agonist (high dose) v.vehicle 20-35 min p < 0.05

FIG. 3 shows a bar graph depicting the urine output of mice comparingICI204448 to Compound 1. The resultant diuresis shown in FIG. 3 in mLurine collected over six hours has been normalized for mouse weight (mLper 100 g body weight). The p value comparing Compound 1 high dose withlow dose IC1204448 was p=0.57. The p value comparing Compound 1 highdose with high dose IC1204448 was p=0.042. The trend of Compound 1 wasnot suggestive of a diuretic effect. The normalized urine volumescollected for the kappa agonist were found to be consistent with priorstudies investigating the overall kappa effect (See e.g., Barber et al,Br. J. Pharmacol., (1994) 111, 843-851).

Example 2—Arthritis Model of Pain in Rats

This study evaluated the efficacy of a single intraperitoneal injectionof Compound 1 on hyperalgesic nociceptive behaviors in an CFA (CompleteFreund's Adjuvant) Model of Rheumatoid Arthritis Pain in Rats.

Rats have been used as a reliable animal model for the study of pain dueto many similarities of the peripheral and central nervous systems ofrats and humans. These similarities are evident both in terms ofbehavioral responses to painful conditions and in terms of painrelieving effects of various therapeutic agents (i.e. opiates andnonsteroidal anti-inflammatory drugs) in both species.

Methods Animal Selection

A statistical power calculator (Massachusetts General Hospital on-linepower calculator, http://hedwig.mgh.harvard.edu/sample_size/size.html)was used to determine the appropriate group size to ensure interpretableand reproducible results. Data from previous studies were input into thecalculator and the group size was calculated based on a jointcompression threshold difference of 68 g with a power of 80% and astandard deviation of 51 g. These parameters resulted in a group sizecalculation of 10.

A total of 55 rats were treated with CFA to ensure that at least 50 rats(i.e., ten rats for each of five groups) met the inclusion criterion. Ithas been established that intracapsular injection of CFA into the anklejoint leads to a robust pain state that can be characterized bymechanical hyperalgesia in approximately 90% of rats (that is, 10% ofthe rats undergoing CFA injection do not meet the study inclusioncriterion for mechanical hyperalgesia). Therefore, to ensure that 50rats meet inclusion criteria, 55 animals were injected with CFA assuggested by the power analysis.

Animal Testing

Following IACUC approval and acclimation, inflammatory arthritis painwas induced in in 55 male, Sprague-Dawley rats by intracapsularinjection of 50 μL of 100% complete Freund's adjuvant (CFA) into thetibio-tarsal joint of the left hind leg. Mechanical hyperalgesia wasassessed via joint compression thresholds (JCTs) JCTs were determinedprior to CFA injection and 14 days post-CFA, prior to study articleadministration. At that time, 50 animals that met the inclusioncriterion were randomly assigned to 5 groups with 10 animals per group(Table 3). To further confirm the validity as a model for arthriticpain, the anti-inflammatory cyclooxygenase-2 inhibitor, celecoxib, wasused as an active control.

TABLE 3 Study Design Test System ID: Rat: Sprague-Dawley: Male Species:Breed: Sex Dose Day of Group Dose Vol. Admin./ # Treatment N (mg/kg)(mL/kg) Route Frequency 1 Vehicle 10 NA 5 IP Day 0/1× (Ethanol:Tween80:Normal Saline - 1:1:8) 2 Compound 1 10 1 5 IP Day 0/1× 3 Compound 110 5 5 IP Day 0/1× 4 Compound 1 10 10 5 IP Day 0/1× 5 Celecoxib 10 30 5PO Day 0/1×

Animals were administered a single dose of test or control compound onday 0 (i.e., 14 days after administration of CFA) and thresholds weredetermined 1, 2, and 4 hours after compound administration JCTs in testcompound-treated animals were compared to those in vehicle-treatedanimals to determine the analgesic efficacy of the test compound. Allbehavioral evaluations were performed by a blinded observer.

Mechanical hyperalgesia was measured using a digital Randall-Selittodevice (dRS; IHTC Life Sciences©; Woodland Hills, Calif.; see Randall,L. O., and J. J. Selitto “A Method for Measurement of Analgesic Activityon Inflamed Tissue.” Arch. Int. Pharmcodyn. 11 (1957): 409-19). Animalswere allowed to acclimate to the testing room for a minimum of 15minutes before testing. Animals were placed in a restraint sling thatsuspended the animal, leaving the hind limbs available for testing. Thestimulus was applied to the ankle joint by a blunt tip and pressure wasapplied gradually over approximately 10 seconds Joint compressionthreshold values were recorded at the first observed nocifensivebehavior (vocalization, struggle, or withdrawal). One reading per jointwas taken at each time point, and a maximum stimulus cutoff of 500 gramswas used to prevent injury to the animal. The mean and standard error ofthe mean (SEM) were determined for ipsilateral and contralateral jointsfor each treatment group at each time point.

After the pre-dosing baseline assessment on Day 0, only animals thatexhibited at least a 25% decrease in joint compression thresholds (JCTs)from pre-injury baseline to pre-dosing baseline were included in thestudy. All testing was performed in a blinded manner, with allexperimenters involved in the study being unaware of the groupassignment of any animal they were testing. Animals were assigned totreatment groups based on Day 0 pre-dosing JCTs so that group means ofthe ipsilateral JCTs were approximately equal. Animals were ranked byipsilateral JCT and treatments assigned randomly within stratifiedsub-groups according to the total number of treatment groups in thestudy. The volume of test or control article injected was 5 mL/kg. Theanimals were dosed in sequence based on animal number so that thedistribution of treatment across a given set of animals was notpredictable.

Results Mechanical Hyperalgesia Development.

To verify the development of mechanical hyperalgesia due to CFA-inducedrheumatoid arthritis pain, ipsilateral and contralateral jointcompression thresholds (JCTs) were assessed prior to CFA injection,pre-dosing on Day 0, and 1, 2, and 4 hours post-dosing. Ipsilateral JCTswere compared to contralateral JCTs using an unpaired t-test at eachtime point as shown in FIG. 4 and Table 4, below. As shown in FIG. 4,Ipsilateral JCTs were significantly lower at all post-CFA time points,indicating persistent mechanical hyperalgesia due to CFA injection.

FIG. 4 shows the mean±standard error of the mean (SEM) values foripsilateral and contralateral joint compression thresholds (JCTs) invehicle-treated animals. All animals received 5 mL/kg vehicle ([1 part]Ethanol: [1 part] Tween 80. [8 parts] normal 0.9% Saline) viaintraperitoneal injection (n=10). Ipsilateral JCTs were significantlylower at all post-CFA time points, indicating persistent mechanicalhyperalgesia due to CFA injection (pre-dose baseline and 1-hour,p<0.001; 2-hour and 4-hour, p<0.0001 vs. contralateral).

To further verify the development of mechanical hyperalgesia due to CFAinjection, a repeated-measured one-way ANOVA was performed onipsilateral JCTs across all time points tested (Table 4). All post-CFAJCTs were significantly higher than at pre-CFA, indicating significantand persistent mechanical hyperalgesia due to CFA injection.

TABLE 4 Development of Mechanical Hyperalgesia—Statistical TableUnpaired t-test, two-tailed, Ipsilateral vs. Contralateral Time Point tDf p-Value Pre-Injury Baseline 1.22 18 0.240 Pre-Dosing Baseline 4.11 180.0007 1 Hour 4.66 18 0 0002 2 Hour 5.98 18 <0.0001 4 Hour 5.56 18<0.0001

Test Article Assessment:

Fourteen days after CFA injection, on Day 0, mechanical hyperalgesia wasassessed at the pre-dosing baseline (prior to test and control articleadministration) and 1, 2, and 4 hours post-dosing with test and controlarticles. Animals were given an intraperitoneal injection of eithervehicle, or Compound 1, or an oral gavage dose of celecoxib. All threedoses of Compound 1 (1, 5, and 10 mg/kg) significantly reversedmechanical hyperalgesia at the 2-hour and 4-hour time points, while the10 mg/kg dose significantly reversed mechanical hyperalgesia at allthree time points tested (1, 2, and 4 hours post dose) as shown in FIG.5.

FIG. 5 shows the mean±SEM for ipsilateral paw compression thresholdsfollowing CFA injection in animals treated with either vehicle (5 mL/kg,IP), Compound 1 (1, 5, or 10 mg/kg, intraperitoneal (IP)), or celecoxib(30 mg/kg by mouth (PO)) Ten rats were evaluated in each group. Compound1 (10 mg/kg) significantly increased paw compression thresholds comparedto vehicle at all time points, (p<0.001, one-way ANOVA) and the otherdoses of Compound 1 (1 mg/kg and 5 mg/kg) significantly improved pawcompression thresholds at both the 2-hour and 4-hour time points(p<0.01, one-way ANOVA). Celecoxib did not significantly improvethresholds at 1-hour but did show improvement compared to vehicle at the2-hour and 4-hour time points (p<0.001, t test).

As shown in FIG. 5, intraperitoneal administration of Compound 1significantly reversed CFA-induced mechanical hyperalgesia. At 1 mg/kgand 5 mg/kg, Compound 1 significantly increased JCTs at the 2- and4-Hour time points. At 10 mg/kg, Compound 1 significantly increased JCTsat all three post-dosing time points tested (1-, 2-, and 4-Hour). Thereversal in mechanical hyperalgesia was comparable to the activecontrol, celecoxib.

Example 3—Neuropathic Pain Model in Rats

This study evaluated the efficacy of a single intraperitoneal injectionof Compound 1 and the comparator, gabapentin, in the spinal nerveligation (SNL) model for neuropathic pain in the rat. Rats have beenused as a reliable animal model for the study of pain due to manysimilarities of the peripheral and central nervous systems of rats andhumans. These similarities are evident both in terms of behavioralresponses to painful conditions and in terms of pain relieving effectsof various therapeutic agents (i.e. opiates and nonsteroidalanti-inflammatory drugs) in both species. Further, rats are vertebrates,which is necessary when investigating the effects of neuropathic pain.

Methods Animal Selection

A statistical power calculator (Massachusetts General Hospital on-linepower calculator, http://hedwig.mgh.harvard.edu/sample_size/size.html)was used to determine the appropriate group size to ensure interpretableand reproducible results. Data from previous studies were input into thecalculator and the group size was calculated based on a thresholddifference of 4.5 grams with a power of 80% (mean control=3.42, meantreated=7.92, standard deviation=2.1). These input data resulted in agroup size calculation of 10.

A total of 55 rats were used to ensure that at least 50 rats (i.e., tenrats for each of five groups) met the inclusion criteria. It has beenestablished that ligation of the L5 and L6 spinal nerves leads to arobust pain state, characterized by tactile allodynia and mechanicalhyperalgesia in approximately 90% of rats (that is, ˜10% of the ratsundergoing SNL surgery do not meet the study inclusion criteria formechanical sensitivity). Therefore, to ensure 50 rats met inclusioncriteria (as indicated by the power analysis), surgery was performed on55 animals.

Animal Testing

Following IACUC approval and acclimation, neuropathy was induced in 55male, Sprague-Dawley rats by surgically ligating the 5^(th) and 6^(th)lumbar spinal nerves (L5 and L6), a procedure also known as spinal nerveligation (SNL). Mechanical sensitivity was assessed via paw compressionthresholds using a digital Randall-Selitto device. Thresholds weredetermined prior to surgery and 15 days post-surgery, prior to studyarticle administration. At that time. 50 animals that met the inclusioncriteria were assigned to 5 groups with 10 animals per group (Table 5)To further confirm the validity as a model for neuropathic pain,gabapentin was used as an active control.

TABLE 5 Study Design Test System ID: Rat: Sprague-Dawley: Male Species:Breed: Sex Dose Day of Group Dose Vol. Admin./ # Treatment N (mg/kg)(mL/kg) Route Frequency 1 Vehicle 10 NA 5 IP Day 0/1× (Ethanol:Tween80:Normal Saline - 1:1:8) 2 Compound 1 10 1 5 IP Day 0/1× 3 Compound 110 5 5 IP Day 0/1× 4 Compound 1 10 10 5 IP Day 0/1× 5 Gabapentin 10 1005 IP Day 0/1×

Animals were administered a single dose of test or control compound onday 0 (i.e., 15 days after SNL) and thresholds were determined 1, 2, and4 hours after compound administration. Response thresholds in testcompound-treated animals were compared to those in vehicle-treatedanimals to determine the analgesic efficacy of the test compound. Allbehavioral evaluations were performed by a blinded observer.

Mechanical hyperalgesia was measured using a digital Randall-Selittodevice (dRS, IITC Life Sciences©; Woodland Hills, Calif.) (see Randall,L. O., and J. J. Selitto “A Method for Measurement of Analgesic Activityon Inflamed Tissue.” Arch. Int. Pharmcodyn. 11 (1957): 409-19). Animalswere allowed to acclimate to the testing room for a minimum of 15minutes before testing. Animals were placed in a restraint sling thatsuspends the animal, leaving the hind limbs available for testing. Thestimulus was applied to the plantar surface of the hind paw by acone-shaped tip and pressure was applied gradually over approximately 10seconds. Paw compression threshold values were recorded at the firstobserved nocifensive behavior (vocalization, struggle, or withdrawal)One reading per paw was taken at each time point, and a maximum stimuluscutoff of 300 grams was used to prevent injury to the animal. The meanand standard error of the mean (SEM) were determined for ipsilateral andcontralateral paws for each treatment group at each time point.

After the pre-treatment baseline assessment on day 0, only animals thatexhibited at least a 25% decrease in thresholds from pre-injury baselineto pre-dosing baseline OR a 1.5 ratio of contralateral/ipsilateralthresholds were included in the study. All testing was performed in ablinded manner, with all experimenters involved in the study beingunaware of the group assignment of any animal they were testing.

Animals were assigned to treatment groups based on Day 0 pre-dosing dRSpaw compression thresholds so that group means of the ipsilateral pawcompression thresholds were approximately equal. Animals were ranked byipsilateral paw compression threshold measurement from lowest to highestand treatments assigned randomly within stratified sub-groups accordingto the total number of treatment groups in the study.

The volume of test or control article injected was 5 mL/kg. The animalswere dosed in sequence based on animal number, so that the distributionof treatment across a given set of animals was not predictable.

Results Hyperalgesia Development:

In order to verify the development of mechanical hyperalgesia due to SNLsurgery, ipsilateral and contralateral paw compression thresholds wereassessed prior to SNL surgery, post-SNL surgery prior to day 0 dosing,and at 1, 2, and 4 hours post-dosing on day 0. Ipsilateral pawcompression thresholds were compared to contralateral paw compressionthresholds using an unpaired t-test at each time point ipsilateral pawcompression thresholds were significantly lower at all post-SNL timepoints as shown in FIG. 6 and Table 6, indicating persistent mechanicalhyperalgesia due to SNL surgery.

FIG. 6 shows the mean±standard error of the mean (SEM) values foripsilateral and contralateral paw compression thresholds following SNLsurgery in vehicle-treated animals. All animals received vehicle ([1part] Ethanol:[1 part] Tween 80. [8 parts] normal 0.9% Saline—5 mL/kg)via intraperitoneal injection (n=10). Significantly reduced ipsilateralpaw compression thresholds were noted at all time points followinginjury: Pre-dosing baseline (p<0.001), I-hour (p:0.0001), 2-hour(p<0.001) and 4-hour (p<0.001) vs contralateral.

To further verify the development of mechanical hyperalgesia due to SNL,a repeated-measured one-way ANOVA was performed on ipsilateral pawcompression thresholds (PCTs) across all time points tested (Table 6).All post-SNL PCTs were significantly higher than at pre-SNL, indicatingsignificant and persistent mechanical hyperalgesia due to SNL

TABLE 6 Development of Hyperalgesia—Statistical Table Unpaired t-test,two-tailed, Ipsilateral vs. Contralateral Time Point t Df p-ValuePre-Injury Baseline 0.6803 18 0.505 Pre-Dosing Baseline 4.59 18 0.0002 1Hour 6.542 18 <0.0001 2 Hour 4.098 18 0.0007 4 Hour 7.304 18 <0.0001

Test Article Assessment:

Fifteen days after SNL surgery, on study day 0, mechanical hyperalgesiawas assessed at the pre-dosing baseline (prior to test and controlarticle administration) and 1, 2, and 4 hours post-dosing with test andcontrol articles. Animals were given an intraperitoneal injection ofeither vehicle ([1 part] ethanol, [1 part] Tween 80, [8 parts] normal0.9% saline). Compound 1, or Gabapentin. The 1 mg/kg dose of Compound 1did not significantly reverse mechanical hyperalgesia at any of the timepoints tested. The 5 mg/kg and 10 mg/kg doses of Compound 1 did notsignificantly reverse mechanical hyperalgesia at the 1-hour post-dosingtime point but did significantly reverse mechanical hyperalgesia at the2- and 4-hour post-dosing time points as shown in FIG. 7.

FIG. 7 shows the mean error of the mean (SEM) for ipsilateral pawcompression thresholds following SNL surgery in vehicle-, gabapentin-,and Compound 1-treated animals. All animals received vehicle ([1 part]ethanol, [1 part] Tween 80, [8 parts] normal 0.9% saline—5 mL/kg),Compound 1 (1, 5, or 10 mg/kg) or gabapentin (100 mg/kg) viaintraperitoneal injection (n:=10/group).

The test compound assessed in this study, Compound 1, was administeredat doses of 1 mg/kg, 5 mg/kg, and J0 mg/kg. The 1 mg/kg dose did notsignificantly reverse SNL-induced mechanical hyperalgesia at any timepoint tested. The 5 mg/kg and 10 mg/kg doses did not significantlyreverse SNL-induced mechanical hyperalgesia at the 1-hour post-dosingtime point but did significantly reverse SNL-induced mechanicalhyperalgesia at the 2-hour (p<0.05 versus vehicle by one-way ANOVA) and4-hour (p<0.001 versus vehicle by one-way ANOVA) post-dosing time pointsGabapentin significantly reversed SNL-induced mechanical hyperalgesia at1, 2, and 4-hours (p<0.01 versus vehicle by t test). The reversal ofmechanical hyperalgesia by Compound 1 at 5 mg/kg and 10 mg/kg did notdiffer significantly from the active control, gabapentin.

Example 4—Bone Cancer Model of Pain in the Rat

In this study, the effect of test article Compound 1 on osteolytic bonecancer pain induced in the MRMT-1 model was studied in female,Sprague-Dawley rats. This study evaluated the efficacy of a singleintraperitoneal injection of Compound 1 and the comparator, subcutaneousmorphine, in the MRMT-1 cancer cell model of osteolytic cancer pain inthe rat. Rats have been used as a reliable animal model for the study ofpain due to many similarities of the peripheral and central nervoussystems of rats and humans. These similarities are evident both in termsof behavioral responses to painful conditions and in terms of varioustherapeutic agents (e.g., opioids, non-steroidal anti-inflammatorydrugs, anticonvulsants and antidepressants) in both species. Rats areamong the best species for determining the predictability of efficacy oftherapeutic agents in humans. Further, rats are vertebrate animals whichenables the investigation of the effects of post-surgical pain.

Methods Animal Selection

A statistical power calculator (Massachusetts General Hospital on-linepower calculator, http://hedwig.mgh.harvard.edu/sample_size/size html)was used to determine the appropriate group size based on a thresholddifference of 14 percent as measured by weight bearing score (WBS) witha power of 90% and a standard deviation of 9 (parallel study with aquantitative measurements). These parameters and input data fromprevious studies resulted in a group size calculation of 10 animals pergroup.

Originally, five experimental groups were proposed as follows: Group 1(vehicle), Group 2 (1 mg/kg Compound 1); Group 3 (5 mg/kg Compound 1);Group 4 (10 mg/kg Compound 1); and Group 5 (morphine). However, thecancer model was initially successfully induced in only 40 animals,which would have resulted in only eight animals per group. Moreover, dueto considerable variability in the vehicle group, at interim evaluationthe positive control (morphine) did not significantly reduce painbehaviors at any time point. Accordingly, in order to ensurereproducible results, the experiment was redesigned. A new poweranalysis was performed yielding a group size of 14 to enable a detectionof 11.5 units (percent WBS) with a standard deviation of 9 units at apower of 90%. The IACUC approved an additional 6 animals in each of 3groups (vehicle (Group 1), Compound 1 [10 mg/kg] (Group 4) and morphine(Group 5). Only 15 of the additional 18 animals developed the conditionresulting in successful model development with 13 animals per group.

Animal Testing

Osteolytic bone cancer was produced by an injection of 3000 mammarygland carcinoma cells (MRMT-1) into the intramedullary space of thetibia. Animals received either vehicle or Compound 1 (10 mg/kg)intraperitoneally on Day 0. Morphine (6 mg/kg) served as the positivecontrol for this study and was administered via subcutaneous injection.Bone cancer pain was assessed by measuring hind limb percent weightbearing scores (percent WBS) prior to inoculation (study day −21), andprior to administration (BL), 1, 2, and 4 hours after administration onDay 0.

Hind limb weight bearing scores (WBS) are measured using a LintonIncapacitance Tester (Stoelting Co.©; Wood Dale, Ill.; see Medhurst, S.J., K. Walker, M. Bowes, B. L. Kidd, M. Glatt, M. Muller, M.Hattenberger, J. Vaxelaire, T. O'Reilly, G. Wotherspoon, J. Winter, J.Green, and L Urban. “A Rat Model of Bone Cancer Pain” Pain 96 (2002).129-40). Animals were allowed to acclimate to the testing room for aminimum of 15 minutes before testing. Animals are placed in an acrylictest chamber. When the animal is in the correct position in the testchamber an evaluation of force was taken, with the evaluation measuringthe average force exerted individually by each hind paw over a threesecond interval Three evaluations of force per animal are taken at eachtime point. The percent WBS for the injured leg is calculated for eachevaluation of force using the following formula.

${\%{weight}{bearing}{score}} = \text{ }{\left\lbrack \text{⁠}{\frac{{weight}{on}{left}{leg}}{\left( {{{weight}{on}{left}{leg}} + {{weight}{on}{right}{leg}}} \right)}} \right\rbrack \times 100}$

The mean of the 3% WBS values is taken as the % WBS for that time point.The mean and standard error of the mean (SEM) are determined for eachtreatment group at each time point.

Success Criteria

Model creation. Significant decrease in % WBS

Model sensitivity: Significant reversal oft % WBS by morphine.

Inclusion: Only animals that exhibit a post-injury % WBS equal to orless than 40 were included in the study.

Blinding. All testing was performed in a blinded manner, with allexperimenters involved in the study being unaware of the groupassignment of any animal they were testing.

Group assignment. Animals were assigned to treatment groups based on Day0 pre-dosing percent WBS so that group means were approximately equalAnimals were ranked by percent WBS from lowest to highest and treatmentsassigned randomly within stratified sub-groups according to the totalnumber of treatment groups in the study.

Dosing: The volume of test or negative control article injected was 5mL/kg via intraperitoneal injection or 2 mL/kg via subcutaneousinjection for morphine. The animals were dosed in sequence based onanimal number, so that the distribution of treatment across a given setof animals was not predictable (Table 7)

TABLE 7 Study Design Test System ID: Species: Breed: Sex Rat:Sprague-Dawley: Female Dose Dose Vol. Day of Admin./ Group # Treatment N(mg/kg) (mL/kg) Route Frequency 1 Vehicle 13 NA 5 IP Day 0, 1× 2 (notCompound 8 1 5 IP Day 0, 1× evaluated) 1 3 (not Compound 8 5 5 IP Day 0,1× evaluated) 1 4 Compound 13 10 5 IP Day 0, 1× 1 5 Morphine 13 6 2 SQDay 0, 1×

Results

To assess the presence of weight bearing asymmetry throughout thepharmacological assessment period, hind limb weight bearing scores frompre-treatment baselines on Day 0 in the vehicle group were compared tothe pre-inoculation baseline using an un-paired, two-tailed t-test.

Mean hind limb weight bearing scores pre-dosing on Day 0 (BL) weresignificantly lower than pre-inoculation baseline as shown in FIG. 8,indicating the presence of significant weight bearing asymmetry, butonly at pre-dosing baseline Variability in the vehicle group at latertime points was noted.

FIG. 8 shows the mean±standard error of the mean (SEM) values forpercent WBS in vehicle-treated animals during the pharmacologicassessment period. All animals received vehicle (5 mL/kg) viaintraperitoneal injection (n=13). Significant asymmetry inweight-bearing was noted at the pre-dosing time point (p<0.0001 vs.pre-injury, unpaired, two-tailed t-test).

Inoculation with MRMT-1 cancer cells produced, once established, arobust and consistent hind limb weight bearing asymmetry demonstrated bysignificant differences in percent WBS between pre-inoculation andpre-dosing percent WBS in the vehicle group.

Subcutaneous administration of morphine (6 mg/kg) produced atime-dependent reversal of hind limb weight bearing asymmetry at 1 and 2hours when compared to pre-dosing baseline.

Intraperitoneal administration of Compound 1 (10 mg/kg) producedsignificant reversal of hind limb weight bearing asymmetry at 4 hourswhen compared to pre-dosing baseline as shown in FIG. 9.

FIG. 9 shows the mean±standard error of the mean (SEM) values for theweight bearing scores (%) following bone cancer development in vehicle-,morphine-, and Compound 1-treated animals. All animals received vehicle([1 part] ethanol, [1 part] Tween 80, [8 parts] normal 0.9% saline—5mL/kg), or Compound 1 (10 mg/kg) via intraperitoneal injection, ormorphine (6 mg/kg) subcutaneously (n=13/group).

Morphine produced significant change in the weight-bearing asymmetrycompared to vehicle only at 1-hour (p=0.02) Compared to pre-dosebaseline, however, morphine significantly improved weight-bearing at1-hour (p<0.01) and 2-hour time points (p<0.01). Morphine did notproduce significant improvement at 4-hours (p=ns). Compound 1 (10 mg/kg)did produce significant improvement in weight-bearing at 4-hourscompared to pre-dose 131. (p<0.001) (two-tailed t-tests).

EQUIVALENTS

While the present invention has been described in conjunction with thespecific embodiments set forth above, many alternatives, modificationsand other variations thereof will be apparent to those of ordinary skillin the art. All such alternatives, modifications and variations areintended to fall within the spirit and scope of the present invention.

1. A method of treating pain caused by inflammation in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of Compound 1:

or a pharmaceutically acceptable salt, prodrug, solvate, hydrate,tautomer, or isomer thereof.
 2. The method of claim 1, wherein the painis caused by initiation of the inflammatory response.
 3. The method ofclaim 1, wherein the pain is associated with hyperalgesia.
 4. The methodof claim 1, wherein the pain is chronic pain or subacute pain.
 5. Themethod of claim 1, wherein the chronic pain is arthritis pain, low backpain, neuropathic pain, visceral pain, pain due to cancer, pain due toinjury, pain due to joint inflammation, pain due to back disorders, orneck pain.
 6. The method of claim 5, wherein the pain due to cancer iscaused by cancer involving intraperitoneal abdominal and pelvic organsor bone cancer or bone metastases.
 7. The method of claim 5, wherein thepain due to injury is caused by bone, ligament, or tendon injury.
 8. Themethod of claim 1, wherein Compound 1 reduces pain to a similar degreeas a central-nervous system-acting opioid.
 9. The method of claim 8,wherein the central-nervous system-acting opioid activates a mureceptor.
 10. The method of claim 8, wherein the central-nervoussystem-acting opioid is morphine.
 11. The method of claim 1, whereinadministrating Compound 1 does not result in any central-nervous systemside effects.
 12. The method of claim 11, wherein the central nervoussystem side-effects are addiction, constipation, sedation, impairedmentation, somnolence, respiratory depression, nausea, dysphoria, orseizures.
 13. The method of claim 12, wherein administering Compound 1does not result in constipation.
 14. The method of claim 12, whereinadministrating Compound 1 does not result in addiction.
 15. The methodof claim 1, wherein Compound 1 results in synergistic activation ofkappa and delta opioid receptors.
 16. The method of claim 16, whereinthe synergy results from allosteric modulation of kappa receptors bydelta receptor activity.
 17. The method of claim 1, whereinadministration of Compound 1 is similar or superior to a kappa receptoragonist for treatment of acute pain.
 18. The method of claim 1, whereinadministration of Compound 1 is similar or superior to a kappa receptoragonist for treatment of hyperalgesia. 19.-24. (canceled)
 25. Apharmaceutical composition comprising Compound 1:

and a pharmaceutically acceptable carrier.